Device for quantitatively determining the concentration of metals in body fluids

ABSTRACT

This invention provides a measuring method and a device suitable for the quantitative analysis of metal elements contained in body fluids comprising: 
     using the flow injection method for reacting a sample with a reagent in a tubule and analyzing the reacted solution. In essence, the present invention provides; 
     a method for introducing the protein liberated by the reaction of body fluid sample and protein-release reagent into a separating membrane for preventing the passage of protein to separate and remove the liberated protein, and then introducing the reacted solution into a quantitative analysis means for determining and measuring the concentration of target metal contained in the body fluid; and 
     a quantitative analysis device for performing the foregoing method.

This is a continuation of co-pending application Ser. No. 08/081,617filed on Jun. 23, 1993.

BACKGROUND OF THE INVENTION

This invention relates to a method and a device suitable for thequantitative analysis of metal elements contained in body fluids. Thepractice of quantitatively analyzing metal elements contained in bodyfluids has been increasing in importance in the field of clinicalmedicine, and clinical examinations. For example, zinc exists at 0.9 to1.1 ppm in the blood serum of healthy men, but it is reported that alack of zinc causes impairment of the gustatory sense, a decrease inreproductive functions, and growth retardation.

For example, as metal ingredients in the blood are dialyzed duringdialysis, adverse reactions such as complications from zinc deficiencytend to develop rather easily. Therefore, determining the level of zincin blood serum of patients will prove helpful in detecting zincdeficiency, and averting any complications resulting from saiddeficiency. It is preferable that this quantitative analysis beperformed immediately after collection at a medical institute whichcollects body fluids. In actuality, however, most of the body fluidscollected are sent outside for analysis, because the conventional methodrequires skilled operations, and it takes a long time to get results.

In order to effectively determine the concentration of metal elements inbody fluids, it is necessary to remove any proteins from the sampleprior to analyzing it. This is necessitated by the fact that proteinsgenerally impede the quantitative analysis of metal elements in bodyfluids. The protein is liberated by adding a deproteinizing agent suchas an acidic solution of trichloroacetic acid, and further separated bycentrifugation. Then, after performing a pre-treatment corresponding toa target metal element, the deproteinized sample is introduced into aquantitative analysis device.

As to prior art, please refer to "Conventional Method (Centrifugationand Absorptiometry) for Analysis of Metals in Body Fluids by T. Makino,M. Saito, D. Horiguchi and K. Kina: Clinical Chimica Acta, Vol. 20,127-135 (1982)".

The following problems are associated with the conventional method,mentioned above:

(1) A large amount of sample of about 0.2 to about 0.5 ml or more isusually required for the analysis.

(2) Since a series of analytical steps from deproteinizing toquantitative measurement are performed by batch system, it takes aconsiderably long time to obtain the results of analysis.

(3) Since the analytical operation is performed in an open system, theprobability of contamination of the body fluid sample during theoperation is greatly increased.

(4) A skilled technician is needed because the analytical operation iscomplicated.

(5) The rate of error in the measurement of the concentration of metalsis great.

On the other hand, the so-called "flow injection method" is known as acontinuous analysis method. This method requires feeding a reagentsolution into a tubule by a non-pulsatory quantitative pump, etc.,injecting a sample solution into the reagent, mixing the reagent and thesample with one another in the tubule, and then introducing the reactedsolution into a quantitative analysis device. In this method, therequired amount of the sample is small; the likelihood of contaminatingthe sample is almost nil because the steps of mixing, reaction, anddilution are performed in the tubule; and an automatic determination ofthe concentration of the target metal element can be performed with highprecision and within a short period of time. Further, since it ispossible to control the dispersion of the sample by adjusting the fluidconditions of the system, this method is suitable for analyzing a largenumber of samples within a short period of time by appropriatelychanging the conditions according to circumstances. Additionally, theassembly and maintenance of the device is easy and inexpensive.

As to prior art, refer to "Conventional Method of Flow InjectionAnalysis by T. Deguchi, R. Takeshita, A. Tanaka and I. Sanemasa: BunsekiKagaku Vol. 37, 247-252 (1988)".

However, direct quantitative analysis of metal elements contained inbody fluids by the flow injection method have not been performed so far.This is because, the centrifugal step for isolating proteins from thesample was thought to be indispensable to this method. Thus this methodwas not deemed suitable for determining the concentration of metalelements in body fluids.

OBJECTS AND SUMMARY OF THE INVENTION

The present invention aims at solving the problems listed in thepreviously mentioned sub paragraphs (1) to (5) relating to conventionalanalysis method.

Further, it is an object of the present invention, to provide a methodand a device which permits quantitatively analyzing trace amounts ofbody fluid samples, in order to determine the concentration of saidtarget metal elements in body fluids as a whole.

It is still, another object of the present invention, to provide amethod and a device for quantitative analysis of body fluid samples inorder to determine the concentration of target metal elements with highaccuracy and in a shorter period of time, by applying the flow injectionmethod.

Briefly, this invention comprises:

(1) dissolving the protein by adding a body fluid sample into a carriersolution containing a protein-release reagent in the tubule of themeasuring system, or

(2) liberating the protein by adding a body fluid sample into a carriersolution containing a protein-release reagent, and separating theliberated protein by a separating membrane mounted on the tubularchannel.

For the quantitative analysis of metal elements contained in bodyfluids, this invention applies the flow injection method, whichgenerally requires reacting a body fluid sample with a reagent in atubule, and analyzing the reacted solution.

In essence, the present invention provides:

a first method for introducing a body fluid sample and a reagent into acarrier solution, reacting both solutions with one another in the tubuleto liberate the protein from the body fluid sample, and introducing thereacted solution into a quantitative analysis means for determining andmeasuring the concentration of metals contained in the body fluid; and

a second method for introducing the protein liberated by said reactionof the body fluid sample and the protein-release reagent into aseparating membrane for preventing the passage of protein to separateand remove the liberated protein, and then introducing the reactedsolution into the quantitative analysis means for determining andmeasuring the concentration of metals contained in the body fluid.

The above, and other objects, features and advantages of the presentinvention will become apparent from the following description read inconjunction with the accompanying drawings, in which like referencenumerals designate the same elements.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of a quantitative analysis device for the firstmethod of the present invention.

FIG. 2 is a schematic of a quantitative analysis device for the secondmethod of the present invention.

FIGS. 3, 4, and 5 are graphs showing the results of test examples 1 and2, respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The constitution of the present invention is explained referring toFIGS. 1 and 2. These figures are schematics of quantitative analysisdevices invented for performing the first method and second method ofthe present invention. The same numerals are designated to the deviceunits having the same functions. As shown in these figures, thedifference between both devices lies in the presence of the separatingmembrane part shown by 7 in FIG. 2.

That is, the reacted solution having passed through a reaction part 6,is either

(1) introduced as is into a quantitative analysis means 9 to determineand measure the concentration of metals according to the first method,or

(2) is introduced into a separating membrane 7, which prevents theprotein from passing through it, and thus separates and removes theliberated protein, followed by introducing the reacted solution into aquantitative analysis means 9 to effectively analyze the concentrationof metals according to the second method.

Both first and second methods start with introducing a protein-releasereagent and a trace amount (usually, 100 μl or so) of body fluid sampleinto a carrier solution, allowing this mixed solution to flow through atubule, followed by reacting the protein-release reagent with the bodyfluid sample in the mixed solution to liberate the protein contained inthe body fluid.

In the step involving the introduction of the body fluid sample into themeasuring system, the protein-release reagent may be added into thecarrier solution containing the body fluid sample, or the body fluidsample may be added into the carrier solution containing theprotein-release reagent. However, the latter is simple and convenient inview of the configuration of the device.

The introduced body fluid sample reacts with the protein-release reagentcontained in the solution and liberates the protein in the tubule. Inthis process, it is preferable to meander the tubule in which saidreacted solution flows or to form a reaction part wound like a coil inthe tubule to secure a sufficient reaction time.

In the case of the first method of the present invention, it ispreferable to employ a combination of protein-solubilizing agent anddeproteinizing agent as a protein-release reagent. In this event, it ispossible to add the deproteinizing agent and then the proteinsolubilizing agent into the carrier solution, or to add simultaneouslythe deproteinizing agent and the protein-solubilizing agent. Further, itis also possible to add the deproteinizing agent alone, depending on thekind of target metal for measurement.

Surface active agents which can be used as a protein-solubilizing agentare: dodecyl sodium sulfate, polyvinyl alcohol, acetyltrimethylammoniumbromide (CTA-Br), polyoxyethylene (10) octylphenyl ether (product name:Triton-X100), polyoxyethylene glycol sorbitan monoalkyl ester, and thelike.

On the other hand, in the case of the second method of the presentinvention, the protein-solubilizing agent is not necessarily required,but the deproteinizing agent is indispensable, as a protein-releaseagent.

General deproteinizing agents such as trichloroacetic acid (TCA) andtetramethylammonium hydroxide (TMAH) can be used for both first andsecond methods.

When TCA is used as a deproteinizing agent, dilute hydrochloric acid,dilute nitric acid, or perchloric acid can be used as a carriersolution. When using TMAH as the deproteinizing agent, dilute sodiumchloride solution or water can be used as the carrier solution. Theconcentration of deproteinizing agent, when used for about 100 μl ofbody fluid sample, is sufficient to be from about 0.1 to about 0.5 mol/lor so in the reacted solution. Since the rate of dispersion andretention time, characteristic of the flow injection method, is high inthe reaction process of said tubule, the body fluid sample is dispersedat a certain rate, and the reaction proceeds at a certain rate in thetubule. Even though the protein from the body fluid sample is not fullyisolated and liberated, the amount of protein so liberated over a givenperiod of time remains unchanged. Therefore the step of measuring anddetermining the concentration of the target metal element should not beadversely affected by the presence of a minuscule amount of unisolatedprotein.

In the first method, the proteins in the body fluid sample precipitatein an acidic environment such as one with a pH below 6.5. Therefore,when TCA is used, pH of the solution is adjusted to a range of about 6.5to about 6.9. It is preferred that TMAH be used because it separates anddissolves the protein under a neutral or alkaline condition at pH 7.0 orhigher.

For example, when a solution of 0.5M TCA in 0.1M hydrochloric acid(deproteinizing agent) is added to 0.5 ml of human serum sample, theprotein is liberated, and the solution becomes turbid. Addition of 0.05Mn-dodecyl sodium sulfate (protein-solubilizing agent) to this sampledissolves most of the protein and produces an almost clear solution inwhich most of the protein dissolves, even though a slight turbidness isobserved. Instead of n-dodecyl sodium sulfate, use of polyvinyl alcohol,acetyltrimethylammonium bromide (CTA-Br), polyoxyethylene (10)octylphenyl ether (Triton-X100), or polyoxyethylene glycol sorbitanmonoalkyl ester likewise produces a clear solution.

In the second method, the reacted solution is channelled through theseparating membrane, which prevents the protein from passing through it.This results in the isolation and liberation of the protein from thebody fluid sample and the production of a clear solution. The separatingmembrane part shown by 7 in FIG. 2, comprises of a separating membrane,which isolates and liberates the protein contained in the body fluidsample by virtue of it having smaller transmitting holes than themolecular diameter of the protein.

This separating membrane fractionates and separates the protein, as thereacted solution passes through it, into an organic phase containing theprotein and a liquid phase. Cellulose membranes such aspolytetrafluoroethylene membrane (PTFE membrane) with a hole diameter of0.45 μm, cellulose acetate membrane with a hole diameter of 0.20 μm, andcellulose nitrate membrane with a hole diameter of 0.45 μm, or anaromatic polyamide membrane with a fractional molecular weight of about20,000, can be employed as a separating membrane. The organic phasecontaining the separated protein is finally, channelled to the outsideof the system.

For example, addition of a solution of 0.5M TCA in 0.1M hydrochloricacid (deproteinizing agent) to 0.5 ml of human serum sample, liberatesthe protein contained in the sample and produces a white turbidsolution. The above solution when passed through a PTFE membrane with ahole diameter of 0.45 μm, separates into a white turbid organic phasecontaining the liberated protein and a clear serum. On the other hand,addition of a solution of 10% TMAH by vol in water (deproteinizingagent) to human serum sample produces a slightly yellowish to clearsolution from which the protein separates, and the separating of thesolution by said PTFE membrane likewise produces a clear serum.

The reacted solution, having passed the process for dissolving theprotein by means of a protein-release agent or for removing the proteinby the membrane separation of said protein, is then introduced into ananalyzing means, which determines and measures the concentration of thetarget metal elements. As for the analyzing means for determining theconcentration of metals in this process, conventional analysis methodsfor liquid samples can be used, including inductively coupled plasmaatomic emission spectrometer (ICP spectrometer), atomic absorptionspectrometer, visible/ultraviolet spectrophotometer, infraredspectrophotometer, fluorophotometer, and liquid-phase chromatograph.

In the determining method (the process for determining and measuring theconcentration of metals in body fluids), a pre-treatment step may beperformed, depending upon the selection of the determining method usedto analyze the concentration of metal elements in the body fluid sample.

For example, when using ICP spectrometry or atomic absorptionspectrometry as the determining method, the sample solution is dilutedby a diluent such as water. When using absorptiometry orfluorophotometry as the determining method, a coloring reagent is addedand made to react with the sample solution. If necessary, the pH of thesolution should also be adjusted. A buffer as a reagent can also beadded depending on the selection of the determining method. If the needarises, plural treatments after deproteinization may also be carriedout.

The selection of absorptiometry as the determining method, has the addedadvantage of ease of operation and convenience. Ordinarily, measuringthe concentration of metals in body fluids by absorptiometry, entailsadding a coloring reagent corresponding to the target metal to thedeproteinized sample solution, allowing for the two to react with eachother, and then introducing the reacted sample into the cell of thespectrophotometer, where the absorbance is measured by irradiating a rayof light with a specific wavelength. Then, the absorbance obtained fromthe standard solution containing the same metal is compared with thatfrom said sample solution to calculate the concentration of the targetmetal contained in the sample solution. Any one of several conventionalcoloring reagents can be used in accordance with the kind of a metal tobe detected.

Additionally, this measuring method suits automation. As an example, itis possible to convert the absorbance obtained by spectrophotometer intoelectric signals and send the signals to the operation of a computer tocompare with the absorbance of standard solution entered into thecomputer previously. Thus, the concentration of the target metal in thesample solution is calculated automatically and displayed.

Referring now to the accompanying figures illustrating the measuringdevice of the present invention, the basic part of this device comprisesmaterials, parts and the like usually used for the flow injectionanalysis. Tubules with an inside diameter of 1 mm or less are used forthe tubular system to permit using a trace amount of sample. Thediameter of the tubule may be changed in part, to permit dispersing thebody fluid sample in the reacted solution while it flows in the tubularchannel. A liquid-feed pump is used to quantitatively feed the solutioninto the measuring system. Since the pulsation of the solution in thesystem causes a reduction in the accuracy of measurement and alters thesensitivity of detection, it is preferable to use a non-pulsative pump,such as a double plunger type of pump, as a liquid-feed pump. Tubulesthemselves, and tubules and other parts forming the tubular channel areconnected by connectors to prevent a leakage of liquid, and it ispreferable that said connected parts are freely provided or removed.

In the examples of devices illustrated in FIGS. 1 and 2, PTEF tubes withan inside diameter of 0.5 to 1.0 mm are used as tubules 10a to 10e, anda double plunger type of pump is used as a liquid-feed pump 11a toprevent the pulsation of solution. An introduction tubular channel 1 forthe protein-release reagent and an introduction tubular channel 2 forthe carrier solution are connected to liquid-feed pump 11a.

As the case may be, both introduction tubular channels 1 and 2 may beused as introduction tubular channels for the protein-release reagent,and these tubular channels may introduce the carrier solution in whichthe deproteinizing agent or the protein-solubilizing agent is dissolved.

Introduction tubular channels 1 and 2 communicate with tubules 10a, 10b,respectively, through liquid-feed pump 11a, join one another afterliquid-feed pump 11a, and connect with an introduction part 5 for thebody fluid sample. Introduction part 5, for example, has a sample loopwith a capacity of 100 μl, and an automatic valve to open and dose saidloop. At the time of measurement, the sample loop has already beenfilled up with the body fluid sample, and a solution of protein-releasereagent (if necessary, containing either the deproteinizing agent orprotein solubilizing agent or both) is introduced into said sample loopby the automatic valve. The reacted solution containing both proteinrelease reagent and body fluid sample is led to a first reaction part 6.First reaction part 6, as an example, is a PTFE tube wound like a coilwith a length of 0.5 m and an inside diameter of 1.0 mm, and while thereacted solution passes first reaction part 6, the proteinreleasereagent reacts with the protein of the body fluid sample to liberate theprotein in the tubule.

In the case of the first method of the present invention, the reactedsolution passing first reaction part 6 is led into a second reactionpart 8 through the tubule, but in the case of the second method, it isled into a membrane separating part 7 through the tubule (as illustratedin FIG. 2). Membrane separating part 7 has a separating membrane withsmaller transmitting holes than the molecular diameter of the protein tobe liberated, to selectively prevent the passage of said protein acrossthe membrane, and the tubular channel of the measuring system ispartitioned by said separating membrane.

As stated above, PTFE membrane with a hole diameter of 0.45 μm,cellulose acetate membrane with a hole diameter of 0.20 μm, cellulosenitrate membrane with a hole diameter of 0.45 μm, or aromatic polyamidemembrane with a fractional molecular weight of about 20,000 can also beused as a separating membrane. As the reacted solution passes throughthe separating membrane part 7, the released protein is prevented frompassing through the separating membrane; the liquid phase from which theprotein is extracted passes through the separating membrane due to itsmolecular diameter, leaving behind an organic phase containing theisolated protein. The organic phase is t channelled to the outside ofthe system by channel 12. While a slight amount of protein (residualprotein) remains in the reacted solution after having passed through theseparating membrane, it is possible to dissolve this residual protein byadding the protein-solubilizing agent.

The reacted solution, in which the protein has been dissolved orremoved, is funneled to a second reaction part 8 through a tubule 10e. Atubular channel 10d for introducing the reagent connects with tubule 10eby a joining part 13. Tubular channel 10d has a liquid-feed pump 11b forfeeding the reagent solution, and a tubular channel 3 for introducing areagent solution connects with liquid-feed pump 11b.

Second reaction part 8, as an example, is a PTFE tube wound like a coilwith a length of 0.5 m and an inside diameter of 0.5 mm. While thereagent solution passes second reaction part 8, the reagent reacts withthe body fluid sample in the tubule.

When determining the concentration of Zinc in a body fluid sample, forexample, 2-(5-bromo-2-pyridylazo)-5-(N-n-propyl-N-(3-sulfopropyl)amino]phenol disodium salt (5-Br-PAPS) is used as a reagent. While the reactedsolution passes second reaction part 8, zinc in the body fluid reactswith said reagent to produce a color developing Zn-5-Br-PAPS complex.Second reaction part 8 connects with a spectrophotometer 9.Spectrophotometer 9 may be a commercially available one. The absorbanceof sample is measured by spectrometer 9 and compared with that ofstandard solution to detect the concentration of the target metal.

Preferably, the opening and closing of liquid-feed pumps 11a, 11b,introduction part 5 for body fluids, and individual tubular channels arecontrolled by computer. Measurement data from the quantitative analyzer9 is converted into electric signals which are then transmitted to acomputer, where the signals are processed automatically.

The devices illustrated in FIGS. 1 and 2 are described above as separateones, but since both devices are identical to one another, with theexception of separating membrane part 7 shown in FIG. 2, it will beeasily understood that the bypassing of tubules before and behindseparating membrane part 7, will permit one device to have bothfunctions as shown in FIGS. 1 and 2.

As mentioned above, according to the quantitative analysis method anddevice of the present invention, only the introduction of a given amountof body fluid sample, together with a reagent, makes it possible tomeasure the concentration of the target metal. Thus, the analysisoperation is very easy and convenient. Furthermore, as the series ofsteps from the initially introducing the body fluid sample todeproteinizing and isolating the protein by the use of the separatingmembrane, and finally analyzing the concentration of metal elements, thelevel of contamination of said sample is greatly reduced, thus yieldinghighly reliable results. Furthermore, the automation of theaforementioned series of steps, makes it possible to analyze a largenumber of samples within a short period of time, and produce morereliable results.

EXAMPLES Embodiment 1

In this embodiment, to explain the first method of the presentinvention, zinc contained in a human fluid sample was determined byselecting human serum as the body fluid; using TMAH (deproteinizingagent) and Triton X-100 (protein-solubilizing agent) as protein-releasereagents; using 5-Br-PAPS as a reagent; and employing a visiblespectrophotometer as an analysis means.

In the measuring system comprising the configuration of deviceillustrated in FIG. 1, a solution of 5% (V/V) TMAH in 0.1M trisodiumcitrate was introduced from introduction tubular channel 1 at a flowrate of 0.75 ml/min; 1% (V/V) polyoxyethylene (10) octylphenyl ether(Triton X-100) was introduced from introduction tubular channel 2 at aflow rate of 0.75 ml/min; and 100 μl of human serum sample wasintroduced from introduction part 5 to dissolve the protein in the humanserum.

A solution of 0.1% (W/V) salicylaldoxime--1% (V/V) polyoxyethylene (10)octylphenyl ether (Triton X-100)--0.5% (W/V) "5-Br-PAPS"--0.5M ammoniumacetate in hydrochloric acid is added as a coloring reagent for zincinto the reacted solution at a flow rate of 1.5 ml/min throughintroduction tubular channel 3.

When introducing the human serum sample, the concentration ofhydrochloric acid in said protein-release reagent solution was adjusted,so that pH of the reacted solution after introduction of the human serumsample was from 8.5 to 9.0.

Absorbance by the Zn-5-Br-PAPS complex formed in the sample solution,after addition of the coloring agent, was measured at a wavelength of560 nm. The concentration of zinc in the sample solution was measured onthe basis of the relation between the concentration of zinc and theabsorbance obtained from the standard solution. Ratio-beamspectrophotometer U-1000 of Hitachi, Ltd. having an optical path lengthof 10 mm was used as a photometer.

Comparison of the results obtained from the present measurement withthose from the conventional analysis method is shown in Table 1. In theconventional analysis method, protein was removed by centrifugation, andthen the concentration of zinc was determined by atomic absorptionspectrometry. Each measurement was performed on six samples, and eachsample was measured three times. The mean values is shown in Table 1.The tubular lengths in the first and second reaction parts are 0.5 m ineither case.

                  TABLE 1                                                         ______________________________________                                        (unit: ppm)                                                                            Method of                                                            Sample No.                                                                             the present invention                                                                         Conventional method                                  ______________________________________                                        1        1.4             1.1                                                  2        1.2             1.2                                                  3        1.1             0.7                                                  4        0.8             0.7                                                  5        1.1             0.8                                                  6        1.4             1.1                                                  ______________________________________                                    

As shown above, the measurement results by the quantitative analysismethod of the present invention correspond well with those of theconventional quantitative analysis method. In addition, the accuracy ofmeasurement by the present method is highly reliable, with an error of5% (C.V. %) when repeating the measurement of the same sample 50 times.The present method permits measuring 20 samples in an hour, and thus agreat number of samples can be measured in a short period of time.

Test Example 1

In this quantitative analysis method of the present invention, theinfluence of the concentration of TMAH as a deproteinizing agent on themeasurement was studied. First, in embodiment 1, while the concentrationof the other reagent was kept constant, a zinc complex was produced bychanging the concentration of TMAH alone between 0 and 10% (V/V), andthe absorbance at a wavelength of 560 nm was measured. The results areshown in FIG. 3. As illustrated, almost constant absorbance was detectedwithin the range of 1.0 to 7.5% (V/V) of TMAH concentration. From theseresults, it has been found that the concentration of TMAH should beadjusted from 1.0 to 7.5% (V/V) to effectively liberate the proteinwhile avoiding the hydrolysis of zinc.

Test Example 2

The influence of the amount of fed liquid and the lengths of first, andsecond reaction parts on the measurement was studied. The amount of fedliquid was increased, while the ratio of the amounts of liquid inintroduction tubular channels 1, 2, and 3 was kept constant. 100 μl ofthe standard solution containing zinc at concentration of 1.14 ppm wasused for the sample solution, and the absorbance at a wavelength of 560nm was measured. While the tubular channel length of the second reactionpart was fixed at 0.5 m, the tubular channel length of the firstreaction part was set at 0, 0.5, or 1 m (A: 0 m, B: 0.5 m, and C: 1 m inthe figure), and the influence of each length on the measurement waschecked. Further, while the tubular channel length of the first reactionpart was fixed at 0.5 m, the tubular channel length of the secondreaction part was set at 0, 0.5, or 1 m (A: 0 m, B: 0.5 m, and C: 1 m inthe figure), and the influence of each length on the measurement waschecked.

The results of the first reaction part are shown in FIG. 4, and those ofthe second reaction part are shown in FIG. 5. From the results shown inFIG. 4, it is noted that, when the length of the first reaction part isset at 0.5 m and the amount of fed liquid by pump 11 is set between 1.7and 3.4 ml/min, the reaction to form a Zn-5-Br-PAPS complex proceedssufficiently, resulting in the measurement of zinc concentration withthe highest sensitivity.

For the second reaction part, even if the intensity of detection in FIG.5 (A) is high, the accuracy of measurement decreases depending onindividual samples. Additionally, if the length of the second reactionpart is set at 0.5 m and the amount of liquid fed by pump 11 is setbetween 1.1 and 3.4 ml/min, the concentration of zinc can be determinedwith the highest sensitivity.

Test Example 3

In order to coordinate the influence of coexisting elements, knownamounts of metal elements and organic substances were added to a humanserum sample, and this sample was introduced into the analytical deviceof embodiment 1 to measure the concentration of zinc. The results areshown in Table 2. Similar to Zinc, it has been found that heavy metalswhich form complexes with 5-Br-PAPS do not affect the measurement ifthese heavy metals exist in usual amounts in the human serum. The sameresults are obtained for organic substances. Iron, however, is thoughtto affect the measurement and analysis of the metal elements in a bodyfluid sample, because its concentration probably increases in the samplesolution, due to hemolysis at the time of collecting samples.

Therefore, adequate care should be exercises in the collection of suchsamples.

                  TABLE 2                                                         ______________________________________                                        Influence of coexisting substances on body fluid sample                       Coexisting Coexistent    Usual concentration                                  substance  concentration in serum solution                                    ______________________________________                                        Cu 2+      3        ppm      0.8 to 1.3                                                                             ppm                                     Fe 2+      5        ppm      0.8 to 2.0                                                                             ppm                                     Fe 3+      5        ppm      0.8 to 2.0                                                                             ppm                                     Ni 2+      0.3      ppm      0.6 to 5.0                                                                             ppb                                     Co 3+      3        ppm      0.04 to 0.3                                                                            ppb                                     Al 3+      3        ppm      2.0 to 6.0                                                                             ppb                                     P          500      ppm      115 to 163                                                                             ppm                                     Mg 2+      1        mg/ml    20       ppm                                     Ca 2+      1        mg/ml    100      ppm                                     Hemoglobin 1        mg/ml                                                     Bilirubin  1        mg/ml                                                     Albumin    50       mg/ml                                                     Trisodium citrate                                                                        No interference                                                    Ascorbic acid                                                                            No interference                                                    EDTA       No coexistence                                                     Sodium fluoride                                                                          10       mg/ml                                                     ______________________________________                                    

Embodiment 2

In this embodiment, to explain the second method of the presentinvention, the concentration of zinc contained in a human fluid samplewas determined by selecting human serum as the sample, using TCA(deproteinizing agent) as a protein-release reagent, using 5-Br-PAPS asa coloring reagent, and employing the visible spectrophotometer as ananalysis means.

In the measuring system comprising the configuration of the device asshown in FIG. 2, a solution of 0.4M TCA in 0.1M hydrochloric acid wasintroduced at a flow rate of 0.75 ml/min from introduction tubularchannel 1; water or dilute hydrochloric acid as a carrier solution wasintroduced at a flow rate of 0.75 ml/min from introduction tubularchannel 2; 100 μl of human serum sample was introduced from introductionpart 5 to liberate the protein contained in the human serum; and theliberated protein was removed by membrane separation.

Successively, as a coloring reagent for zinc, a solution of 0.1% (W/V)salicylaldoxime--1% (V/V) polyoxyethylene (10) octylphenyl ether (TritonX100)--0.5% (W/V) "5Br-PAPS"--0.5M ammonium acetate in hydrochloric acidwas added to the sample solution at a flow rate of 1.5 ml/min. Inaddition, when the human serum sample was introduced, the concentrationof hydrochloric acid in said carrier solution was adjusted, so that pHof the reacted solution could be 8.5 to 9.0 after the introductionthereof.

After introduction of the coloring reagent, the concentration of zinc inthe sample solution was measured by using the same spectrophotometer andin quite the same way as for embodiment 1.

The comparison of the results of the present measurement and those ofthe conventional method is shown in Table 3. The conventional analysismethod is as described in embodiment 1. The tubular channel lengths ofthe first and second reaction parts are 0.5 m in either case.

                  TABLE 3                                                         ______________________________________                                        (unit: ppm)                                                                            Method by                                                            Sample No.                                                                             the present invention                                                                         Conventional method                                  ______________________________________                                        1        1.4             1.1                                                  2        1.2             1.2                                                  3        1.1             0.7                                                  4        0.8             0.7                                                  5        1.1             0.8                                                  6        1.4             1.1                                                  ______________________________________                                    

As shown in the above table, the results of the measurement by thequantitative analysis method of the present invention correspond wellwith those by the conventional quantitative analysis method. Moreover,when repeating the measurement of the same sample 50 times, the accuracyof measurement was high with an error of 5% (C.V. %) as compared withthat of the conventional quantitative analysis method.

According to this analysis method, it is possible to measure 20 samplesin an hour, and thus a great number of samples can be measured in ashort period of time.

Test Example 4

Influence of the concentration of TCA as a deproteinizing agent, on themeasurement of the concentration of metal elements in a body fluidsample, was studied in the same way as for test example 2. First, inembodiment 2, while keeping the concentration of the other reagentconstant, only the concentration of TCA was changed between 0 and 1.25Mto produce a zinc complex, and the absorbance at a wavelength of 500 nmwas measured.

From the results, it has been found that the concentration oftrichloroacetic acid should be adjusted to be 0.05M to 1.0M, preferablyabout 0.4M, to effectively liberate the protein while avoiding thehydrolysis of zinc.

Test Example 5

Influence of the amount of fed liquid as well as the lengths of thefirst and second reaction parts on the measurement was studied in thesame way as for test example 2.

As a result, it has been found that, if the length of the first reactionpart is set to be 0.5 m and the amount of fed liquid by pump 11 is setbetween 1.7 and 3.4 ml/min, the reaction to form a Zn-5Br-PAPS complexproceeds sufficiently, preventing a loss of sensitivity due to thedilution of the sample, and the concentration of zinc can be determinedwith the highest sensitivity.

Further, it has also been found that, if the length of the secondreaction part is set to be 0.5 m, and the amount of fed liquid by pump11 is set between 1.1 and 2.8 ml/min, the concentration of zinc can bedetermined likewise with the highest sensitivity.

Test Example 6

The same measurement as for test example 3 was performed by employingthe analysis device used for embodiment 2, to coordinate the influenceof coexisting elements.

The results are quite identical to those of test example 3 shown inTable 2.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

What is claimed is:
 1. A quantitative flow injection analysis device fordetermining the concentration of metals contained in body fluidscomprising:(a) a source of a protein-release reagent comprising aprotein-precipitating agent; (b) a tubular channel in which a carriersolution containing said protein-release reagent flows; (c) anintroduction part for introducing body fluid sample into said tubularchannel; (d) a first reaction part of said tubular channel in which thebody fluid sample and said protein-release reagent react to formprecipitated protein and a liquid phase containing metals; (e) amembrane separating part located downstream from said first reactionpart for separating, and liberating said protein by preventing thepassage of said protein through said separating membrane; (f) a secondintroduction part for introducing a second reagent into said liquidphase having transmitted the separating membrane; (g) a second reactionpart of said tubular channel in which the metal of said liquid phasereacts with said second reagent to produce a detectable compound; and(h) a measuring part for determining the concentration of metalcontained in said liquid phase previously introduced from said secondreaction.
 2. A quantitative analysis device as claimed in claim 1,wherein said measuring part for determining the concentration of metalis at least one device selected from the group consisting of inductivelycoupled plasma atomic emission spectrometer, atomic absorptionspectrometer, visible/ultraviolet spectrophotometer, infraredspectrophotometer, fluorophotometer, and liquid-phase chromatograph. 3.A quantitative analysis device as claimed in claim 1, wherein saidmeasuring part for determining the concentration of metal is acombination of one or more devices selected from the group consisting ofinductively coupled plasma atomic emission spectrometer, atomicabsorption spectrometer, visible/ultraviolet spectrophotometer, infraredspectrophotometer, fluorophotometer, and liquid-phase chromatograph. 4.A quantitative analysis device as claimed in 1, wherein said separatingmembrane is at least one membrane selected from the group consisting ofpolytetrafluoroethylene membrane, cellulose acetate membrane, cellulosenitrate membrane, and aromatic polyamide membrane.
 5. A quantitativeanalysis device as claimed in claim 4, wherein:saidpolytetrafluoroethylene membrane having a hole diameter of 0.45 μm; saidcellulose acetate membrane having a hole diameter of 0.20 μm; saidcellulose nitrate membrane, having a hole diameter of 0.45 μm; and; saidaromatic polyamide membrane having a fractional molecular weight of20,000.
 6. A quantitative analysis device as claimed in claim 1, whereinsaid protein-precipitating agent is at least one of a trichloroaceticacid and tetramethylammonium hydroxide.
 7. A quantitative analysisdevice as claimed in claim 1, wherein said carrier solution is at leastone member selected from the group consisting of dilute hydrochlorideacid, dilute nitric acid, and perchloric acid.